1. Field of Invention
The present invention generally relates to methods and compositions for the use of tamoxifen and other anti-estrogenic compounds in combination with immune modulator agents (immunoglobulin inhibitors of estrogen responsive cancer cell growth), to treat or prevent breast cancer.
2. Description of Related Art
In 1896, a British physician named Beatson reported that öophorectomy had palliative effects for breast cancer patients (1). In 1905, Lett confirmed this observation with a larger patient trial (2). Clearly ovarian products were either directly or indirectly significant in breast cancer growth. From these earliest clinical observations, chemical and endocrine research continued and culminated in the identification of the primary ovarian/follicular agents responsible. The active agents proved to be a class of cholesterol derived steroid hormones now designated estrogens. In 1929 and 1930, Doisy and colleagues crystallized estrogens including estrone [3-hydroxy-estra-1,3,5(10)-trien-17-one] (E1) from human pregnancy urine (3,12). Estradiol-17β[estra-1,3,5(10)-triene-3, 17β-diol] (E2) was also isolated from sow follicular fluid (4). The remaining major estrogen, estriol [1,3,5-estratriene-3,16α,17β-triol] (E3) has also been defined.
The relative potency of these three hormones is known today to be E2>E1>>>>E3 (5). With regard to breast cancer cell growth, E2 and E1 are in the main considered the most physiologically relevant (6-9). Estriol is most likely relevant during pregnancy when the maternal plasma level is significantly elevated (10). During pregnancy, maternal E3 is formed primarily as a placental conversion product of a steroid produced by the fetal adrenals. Breast cancers are not uncommon during pregnancy (18,22-25). However, all three estrogens are increased in pregnancy (10). In pregnant women, breast cancer is often diagnosed at a later stage (18). It may be that the elevated hormones during this time cause growth of developing breast cancer cells in pregnant females (19). Clearly, however, pregnancy has opposing effects on breast cancer development. On the one hand the increase in hormones can promote cancer cell growth (35). On the other hand, pregnancy and high hormones induce tissue differentiation that ultimately protects the tissue (20,21). Apparently the elevated estrogen levels in pregnancy explain the transient increase in short-term risk of breast cancer following term pregnancy (19). The results of several studies indicate that all three of the estrogenic steroid hormones (i.e. E2, E1 and E3) are important in breast cancer risk in humans (26-28).
The biosynthesis and metabolism of estrogens and estrogen-related steroid hormones has been reviewed (11). The majority of plasma E2 and E1 is synthesized and secreted by cells of the ovarian follicle (29,30). The biochemical synthetic pathway begins with conversion of cholesterol to progesterone, followed by modification of the progestin to form androgens or androgen-like steroids. To form all three types of estrogen, the cholesterol origin “A” ring of “androgens” must be converted to a phenolic structure by the action of aromatases. These key enzymes in the biosynthesis of estrogens are located in the endoplasmic reticulum of ovarian cells.
Estrogens undergo a variety of metabolic transformations including hyroxylations, methylations and reduction. Also, the estrogens are converted to more water-soluble, biologically inactive, glucuronide and sulfate conjugates by the liver. The conjugates are excreted into urine and bile. Earlier studies indicated that estrogen conjugates (e.g. estrone sulfate) might serve as sources of free estrogen in breast cancer cells possessing the appropriate cleaving enzyme(s) to form free steroid (31). More recent work (32-34) indicates this is unlikely, based on tissue culture studies with eight different ER+ cell lines. Estrogen sulfates and glucuronides are cleaved by intestinal flora to regenerate free estrogens that again appear in the plasma and urine via the enterohepatic circulation (36). A high fiber-low fat diet tends to decrease this process. Other intestinal microbial processes also convert inactive estrogen metabolites to active steroid hormones (37). Thus, recycling of estrogens is entirely possible.
However, the sites of synthesis of estrogenic substances in the body are not limited to the ovary (13). While it is understood with premenopausal women that estrogens are primarily of ovarian origin, this is not the case in postmenopausal females (38-41). The question is “what is the origin(s) of estrogens in the postmenopausal female”? This is important because breast cancer rates are much higher in postmenopausal women (42) even though estrogen levels are declining Nonetheless, 80 or 90% of breast cancers in postmenopausal women are ER+ (43), implying they are estrogen growth promoted. This paradox can be explained in part by the suggestion that postmenopausal women with higher risk of developing breast cancer show relatively higher concentrations of endogenous estradiol (44). Also, it is now very clear that adrenal androgenic steroids can be converted to estrogens via the action of aromatases located in mammalian tissues (45). Its activity provides a significant portion of the plasma estrogens even in postmenopausal women (38-41). Aromatase activity has a broad tissue distribution in mammals (45). However, in human women after menopause, adipose tissue is the primary source of endogenous estrogens (46,47). Indeed, obesity is positively correlated with breast cancer (48). Also, aromatase is present in breast tissue and cells and represents an “intracrine” source of stimulating steroid hormone (49). Because of the major role of aromatase in generating breast cancer promoting estrogens in postmenopausal women, a series of aromatase inhibitors has been developed and are now in use as pharmaceutical products or are in and clinical trials as breast cancer treatments (41).
The question of how estrogens regulate target tissue gene expression and growth is of great consequence to this discussion. In 1962, Jensen & Jacobsen (14) came to the conclusion that estrogens acted on sex steroid hormone target tissues via specific cellular receptors. By 1972 to 1974, this research was sufficiently advanced to outline the mechanisms of estrogen action as mediated by an intracellular receptor (15-17). For several years, intense study has proceeded and has been reported in nearly 20 thousand publications (PubMed literature search of “estrogen receptors”). In 1986, the molecular cloning of the original estrogen receptor, now designated ERα, was reported (50,51). This 64-kDalton protein is functionally and structurally related to other receptors and has been classified as a member of the steroid and thyroid hormone superfamily (52). Today, these similar receptors include those for androgens, corticosteroids, progestins, thyroid hormones, vitamin D and retinoic acid.
Although for several years ERα was acknowledged as the only estrogen receptor, variants of it were being identified (55,56). However, in 1995, another type of estrogen receptor, designated ERβ, was cloned from a rat prostate and ovary (57). This initiated a boom of new activity to define the function and properties of ERβ (58,60,61). Indeed, the results suggest that the role of estrogens in male accessory organ function deserves renewed study (58). The characteristics and properties of ERα versus ERβ have been reviewed (58,61,63). For the purposes of this disclosure, it should be noted that the binding affinities of both receptors are approximately equal (61). This was expected. However, one startling fact has surfaced. Mice gene knockout experiments for both ERα (62) and ERβ (60) have confirmed developmental functions for both of these receptors, but have fallen short of providing conclusive evidence that either receptor regulates growth (58). In fact, transfection of ER− cells with a functional ERα led to an estrogen-induced inhibition of cell growth (59). There is a possibility that ERα is a receptor regulating expression of differentiated functions. It is well recognized that growth and differentiation are opposing cell functional states. Differentiated cells divide only slowly if at all. This issue has been reviewed in detail in recent U.S. patent application Nos. 09/852,547 and 09/852,958 and in International Patent Application Nos. PCT/US01/15171 (WO 01/86307) and PCT/US01/15183 (WO 01/85210), also identified in the list of References, below, as items 53 and 54, and hereby incorporated herein by reference). This led to the proposal in those applications that there is another growth regulating estrogen receptor, tentatively designated ERγ (53,54).
The characteristics of ERγ are that it binds estrogens with 10 to 100-fold higher affinities than ERα or ERβ. Furthermore, it is proposed that this receptor is a new gene that is expressed in all estrogen growth responsive target tissues. Data obtained indicate that this receptor is present in eight well-known estrogen responsive tumor cell lines derived from four tissues and three species including human (32-34,53,54).
However, there exist potential alternatives regarding the identity of ERγ. Investigators have cloned two ERα-like “orphan receptors” with unknown functions (64,65). Other forms of estrogen receptors appear to arise as gene product splice variants (58,66). Those with major deletions of the hormone binding domain or the DNA binding domain may be expected to be inactive with respect to estrogen induced growth of breast cancer cells. The function of most of the other types of known variants remains to be established.
Another potentially significant variant has been identified. It is a point mutation that affects the border of the hinge-hormone-binding domains (67). This mutation was found in 34% of a series of 59 specimens of premalignant hyperplasia. Transfection of this mutated ERα caused MCF-7 human breast cancer cells to respond to lower concentrations of estrogen in culture. The full implications of this mutation await more study, but it is clear from the results available at this time, and those presented in the above-identified patent applications (53,54) and other recent publications (32-34), that MCF-7 as well as T47D and ZR-75-1 ER+ breast cancer cells respond to very low concentrations of E2 even without transfection of the mutated ERα. It may be possible that the hypersensitive mutated receptor (67) is present in all ER+ cell types including those from rat mammary and rat pituitary tumors as well as from estrogen-induced kidney tumor cells from Syrian hamster (32-34). This means that a specific mechanism must exist for formation of this receptor in target tissue cells, or that this receptor is derived from a new gene. The latter possibility implies that the response of ER+ cells to very low concentrations of E2 involves the proposed new ERγ (53,54).
The currently available knowledge about estrogen function and estrogen receptors has led to one of the most common treatments for disseminated and/or local ER+ breast cancer, especially in postmenopausal women. Today, selective estrogen receptor modulators (SERMs) are the compounds of choice (68). The mechanism of action of these drugs is to block the growth promoting action of estrogens at the cellular/receptor level, no matter whether the sex steroid hormones are delivered systemically or formed locally in breast tissue via aromatase action on adrenal steroid precursors. Hence, these drugs are classified as anti-estrogens. As a general mechanism of action, anti-estrogens are thought to interfere with the binding of natural estrogens to the growth promoting estrogen receptor(s).
The first potent anti-estrogen developed 1958 was MER-25 or ethamoxytriphetol (76). It then was used to derive clomiphene (77) which is now used to treat amenorrhea. Clomiphene was then modified to give rise to tamoxifen (78). Although several anti-estrogens have been developed, only two are currently FDA approved for treatment of human breast cancer. These are tamoxifen and toremifene. These, and idoxifene and droloxifene, are triphenylethylene derivatives. Notably, the toremifene structure differs from tamoxifen by only a single chlorine atom (69). Since its approval in 1977, tamoxifen has been the SERM of choice for treatment of ER+ breast cancer worldwide (70). Tamoxifen is classified as a “mixed” anti-estrogen because it displays both antagonistic properties (i.e. inhibits breast cancer cell growth) and agnostic properties (i.e. stimulates endometrial cell growth and tumor development) (71).
The action of the anti-estrogens is reversed by lower concentrations of the natural estrogens (53,54). The affinity of tamoxifen for the estrogen receptor is 10 to 100-fold less than that of E2. This is commonly recognized throughout the endocrine cancer field. It is therefore useful to suppress natural estrogens along with application of tamoxifen treatment. This fact is often not recognized clinically. Postmenopausal women are not completely devoid of estrogens. Tamoxifen effectiveness is reduced by residual estrogenic steroid hormones. It is also reduced by the tamoxifen induced elevation of DHEA, E2 and E1 (81-83). This is an unfortunate side effect of using this drug alone.
One of the commonly cited facts concerning tamoxifen is that it acts at cellular sites separate from the estrogen receptor. It is known to influence such cellular activities as protein kinase C as well as several other cellular mechanisms including those related to apoptosis (72). Although non-steroid hormone receptor directed actions are usually considered undesirable, certain very recent co-owned patent disclosures (53,54) describe targeting a non-steroid hormone receptor with new drug combinations whose actions are based on anti-estrogen augmentation/mimicking of the inhibition of growth of ER+ breast cancer cells by the immunoglobulins IgA and IgM of the natural secretory immune system. As described (53,54), the secretory immune system acts as a paracrine negative regulator of ER+ breast cancer cell growth. Employing new serum-free defined culture assay methods (53,54), tamoxifen was shown to mimic the inhibition caused by IgA or IgM in the complete absence of estrogens. This new tamoxifen function represents a clear departure from previous thought concerning how this “mixed function” anti-estrogen acts. Previously, other investigators had reported that tamoxifen inhibited growth factor dependent proliferation of human breast cancer cells in cultures devoid of estrogens and estrogen-like agents (73). However, there was no indication at that time that this anti-estrogen was capable of acting by mimicking the growth inhibitory effects of the natural secretory immune system immunoglobulins IgA, IgM and IgG1.
Another class of anti-estrogens is defined as “pure” because they only affect growth via interaction with estrogen receptors (71). The pure anti-estrogens were discovered about 15 years ago (74). Currently, five compounds are under intense investigation (71). They are abbreviated ICI 164384, ICI 182780, EM-800, RU 58688 and EM-139 (71). Two of these, ICI 164384 and ICI 182780 are in clinical trials. Because tamoxifen resistance develops with time (75), the pure anti-estrogens are thought to be useful as second-line therapies after tamoxifen failure (71). Furthermore, pure anti-estrogens are thought useful because they cause no increase in endometrial cancer (71).
However, the pure anti-estrogens have marked deleterious effects on the cardiovascular and skeletal systems (71), and their usefulness is yet to be established. There remains a need for effective anti-estrogens and for combination therapies of tamoxifen or tamoxifen-like drugs and the “pure” anti-estrogens that may be more effective than either class of drug alone.